Project Summary Fibrosis is a central component of numerous major diseases and is implicated in an estimated 45% of all deaths in the developed world. Hallmarks of fibrosis include myofibroblast (MF) induction, excessive MF proliferation and matrix synthesis, and stiffening or contraction of the extracellular matrix (ECM), but how these changes are interrelated and feedback to reinforce the fibrotic process is not understood. This is in part because we lack a fundamental understanding of how cells sense, mechanically respond to, and in turn alter the structure and mechanics of their surroundings during fibrosis. To fill ths gap in our knowledge and develop treatments that target the mechanisms of fibrosis, we need experimental platforms that allow us to 1) accurately capture the fibrous structure and mechanical behavior of tissues, and 2) monitor the mechanical forces that drive MF differentiation, proliferation, and subsequent fibrotic stiffening. The overall focus of the proposd work is to connect intracellular and extracellular mechanics during the initiation (K99) and reinforcement (R00) of MF induction and proliferation. During the K99 phase, we will develop a technique to measure traction forces in a newly established synthetic ECM that is fibrous, mechanically tunable, and can be reorganized by cells. Using this platform, we will examine whether TGF- induces RhoA-mediated fibril reorganization to increase traction force generation and subsequent MF induction and proliferation. With an understanding of how MF induction and proliferation is initiated, we will go on to examine how increased MF cell density reinforces fibrotic changes during the R00 phase. We will determine whether tension within the fibrillar ECM generated by high densities of contractile MFs spurs further MF induction. Next, we will examine the effect of high MF density on fibrillar network stiffening via ECM synthesis, and query whether this in turn also promotes MF induction. To understand the interplay between the physical surroundings of the cell, intracellular signaling, and cellular traction generation, this work will rely on biomaterial engineering, molecular biology, and finite element modeling approaches. While the applicant has significant experience in biomaterial development and has already established the synthetic fibrillar ECM to be employed in the proposed studies, he requires further training in myofibroblast biology, mechanical modeling, and the development of traction force measurements. The support and training provided by both this award and the assembled mentoring team will grant the applicant tools and expertise critical to his future independent research program. Additionally, this work will shine light on biophysical mechanisms common to fibrotic changes accompanying numerous diseases, and will provide a test bed for therapeutics that can disrupt this process.